RNA Aptamers Selective for TPRV Channels

描述（由申请人提供）：该项目的总体目标是采用一种新的新兴组合化学技术来鉴定和开发以高亲和力和特异性结合所选TRPV通道靶位点的小RNA分子，作为治疗伤害性行为的潜在治疗剂。TRP通道的香草素受体亚家族的两种亚型TRPV 1（辣椒素受体）和TRPV 4（辣椒素受体）显示出对许多伤害性刺激敏感的多模态门控行为。这两种通道广泛分布于背根和三叉神经节的中小型神经元中。然而，鉴定特异性拮抗剂的传统药物发现努力相对缓慢，并且取得了有限的成功，尽管开发努力正在加强。最近发现的小RNA分子可以折叠成独特的3-D结构，以高亲和力和特异性与蛋白质结合结构域结合，类似于单克隆抗体，这为开发新的、强大的一类药物制剂打开了大门。本研究的目的是产生选择性RNA适体，其以高亲和力结合TRPV 1和/或TRPV 4同种型，并作为药理学应用的拮抗剂。为实现这一目标，提出了三个具体目标：1。产生对TRPV通道亚型TRPV 1和TRPV 4具有特异性的高亲和力RNA适体。RNA适体将通过系统进化、扩增和富集高亲和力RNA配体（适体）从大量随机RNA分子中鉴定。2.筛选具有高亲和力的RNA适体作为TRPV亚型的潜在拮抗剂。将评估经鉴定的RNA适体对通过TRPV 1或TRPV 4通道的钙内流的高通量、高含量、动力学筛选，以评价每种经鉴定的适体作为TRPV 1和TRPV 4的功能性拮抗剂的潜力。3.评价所鉴定的RNA适体作为药理学工具/治疗剂的潜在功能。将使用膜片钳分析评价从动力学筛选中鉴定的RNA适体作为药理学工具，以直接评估适体对通道功能的影响。有前途的适体将进一步评估治疗潜力，在体内，在建立模型的TRPV 1和TRPV 4依赖性疼痛行为。这些研究将产生深远的影响，既为鉴定和开发RNA适体作为药理学试剂提供基础，又为伤害性行为的特异性治疗提供新的治疗工具，以及与TRPV离子通道相关的许多其他病理生理学条件。公共卫生相关性：该项目的目标是使用一种新的、新兴的方法来产生小RNA分子（RNA适体，3-D折叠），其以高亲和力和特异性（10; 26）结合与感知疼痛相关的特定通道蛋白（TRPV通道）上的靶位点（40; 57; 61）。选择性阻断TRPV通道功能的RNA分子，因此，疼痛感觉，将被特异性靶向。产生这样的分子将为开发这些和其他化合物作为潜在的药理学工具和治疗剂开辟新的平台，用于治疗与有害刺激或炎症状态相关的广泛的疼痛状况（例如，压力、高温、神经损伤/蛀牙、痛觉过敏和暴露于有毒化学品）（12; 13; 17; 52; 78）。

DESCRIPTION (provided by applicant): The overall goal of the project is to employ a novel, emerging combinatorial chemistry technology to identify and develop small RNA molecules that bind with high affinity and specificity to selected TRPV channel target sites as potential therapeutic agents for treatment of nociceptive behavior. Two isoforms of the vanilloid receptor subfamily of TRP channels, TRPV1 (the capsaicin receptor) and TRPV4 (an osmoreceptor), display polymodal gating behavior with sensitivity to numerous noxious stimuli. Both channels are widely distributed in small- to medium-sized neurons of the dorsal root and trigeminal ganglia. However, traditional drug discovery efforts to identify specific antagonist are relatively slow and have had limited success, although development efforts are intensifying. The recent discovery of small RNA molecules that fold into unique 3-D structures that bind with high affinity and specificity to protein binding domains, akin to monoclonal antibodies, has opened the door to development of a new, powerful, class of pharmacotherapeutic agent. The purpose of the present study is to generate selective RNA aptamers that bind with high affinity to TRPV1 and/or TRPV4 isoforms and act as antagonist for pharmacotherapeutic applications. Three specific aims are proposed to accomplish this goal: 1. To generate high affinity RNA aptamers with specificity for TRPV channel isoforms, TRPV1 and TRPV4. RNA aptamers will be identified from a large pool of random RNA molecules by systemic evolution, amplification, and enrichment of high affinity RNA ligands (aptamers). 2. To screen the high-affinity RNA aptamers for function as potential antagonist of the TRPV isoforms. High throughput, high-content, kinetic screening of identified RNA aptamers on calcium influx through TRPV1 or TRPV4 channels will be assessed to evaluate the potential of each identified aptamer as functional antagonist of TRPV1 and TRPV4. 3. To evaluate the potential function of the identified RNA aptamers as pharmacological tools/therapeutic agents. RNA aptamers identified from the kinetic screens will be evaluated as a pharmacotherapeutic tools using patch clamp analysis to directly assess aptamer effects on channel function. Promising aptamers will be further evaluated for therapeutic potential, in vivo, in established models of TRPV1- and TRPV4-dependent pain behavior. The studies will have far reaching impact both in providing a foundation for identification and development RNA aptamers as pharmacotherapeutic agents, and in providing new therapeutic tools for the specific treatment of nociceptive behavior, as well as for numerous other pathophysiological conditions associated with the TRPV ion channels. PUBLIC HEALTH RELEVANCE: The goal of the project is to use a novel, emerging, approach to generate small RNA molecules (RNA aptamers, 3-D folded) that bind with high affinity and specificity (10; 26) to target sites on specific channel proteins that are associated with sensing pain (TRPV channels) (40; 57; 61). RNA molecules that selectively block the function of the TRPV channels and, therefore, the pain sensation, would be specifically targeted. Generating such molecules would open a new platform for development of these, and other, compounds as potential pharmacological tools and therapeutic agents for the treatment of a broad range of painful conditions associated with noxious stimuli or inflammatory states (e.g., pressure, high temperature, nerve damage/tooth decay, hyperalgesia, and exposure to noxious chemicals) (12; 13; 17; 52; 78).